Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria where they serve as host defense systems, functioning to prevent infection by foreign DNA molecules such as bacteriophage and plasmids that would otherwise destroy or parasitize them. In this defense system, foreign DNA is restricted (cleaved) while host DNA is protected due to modification of the recognition sites by a cognate DNA methyltransferase. This relationship with a DNA methylase ensures that the endonuclease maintains an extremely high degree of specificity. In the course of evolution, any variants with non-cognate restriction activity are subject to strong selection pressure in the form of DNA damage encountered by the bacterial cell.
The remarkable substrate specificity of restriction endonucleases has contributed greatly to the biotechnology revolution. Purification of restriction endonucleases from bacteria allows these enzymes to be used in numerous laboratory applications from gene cloning to mutation detection. Type II restriction endonucleases typically recognize a DNA sequence of 4–8 base pairs (bp). Of the greater than 3000 enzymes characterized so far, 228 distinct substrate specificities have been identified. (Roberts and Macelis, Nucl. Acids Res. 29:268–269 (2001)). The substrate specificity of a restriction endonuclease usually involves single site recognition (e.g. 5′-AGATCT-3′) However, a relatively common feature is recognition of a degenerate sequence. For example, BstYI recognizes 5′-RGATCY-3′ (where R=A or G and Y=C or T). Recognition of a degenerate sequence often limits the utility of a restriction enzyme in laboratory applications since cleavage frequency is excessive. Statistically, an enzyme recognizing a 6-bp sequence cleaves every 4096 bp while an enzyme recognizing 5′-RGATCY-3′ cleaves every 1024 bp on average in a non-biased genome. Restriction endonucleases that cut infrequently (e.g. 8-bp cutting enzymes that cleave every 65,536) are rarely found in nature. Therefore, many engineering efforts are focused on creating less frequent cutters out of existing 6-bp cutters.
More than ten years of endonuclease engineering has resulted in only limited success in altering the substrate specificity of an existing restriction endonuclease. One important example is an attempt to engineer an 8-bp cutting enzyme from EcoRV (5′-GATATC-3′) by using rational protein design based on the high resolution structures of EcoRV complexed with alternate 8-bp substrates (Horton and Perona, J. Biol. Chem. 273:21721–21729 (1998)). In this case, rational protein design pertained to creating one or more specific amino acid substitutions by site-directed mutagenesis of the cloned gene fragment. A conclusion of this effort was that the determinants of altering substrate specificity are difficult to predict even after crystallographic analysis of an endonuclease/DNA substrate complex (Lanio, et al., Protein Eng. 13:275–281 (2000)). The most promising EcoRV variant was derived from a semi-rational approach where twenty-two amino acid residues were chosen for randomization based on examination of the three-dimensional structure. Clones of interest were selected by in vitro analysis of cleavage activity and specificity. From this effort, a triple mutant was identified which exhibited a 25-fold higher rate of cleaving EcoRV sites flanked by AT rather than GC base pairs. (Lanio, et al., J. Mol. Biol. 283:59–69 (1998)).
Many other studies have been conducted to investigate and possibly alter the substrate specificity of the restriction enzymes BamHI (Dorner and Schildkraut, Nucl. Acids Res. 22:1068–1074 (1994), Dorner, et al., J. Mol. Biol. 285:1515–1523 (1999), Whitaker, et al., J. Mol. Biol. 285:1525–1536 (1999), Newman, et al., Science 269:656–663 (1995), Newman, et al., Nature 368:660–664 (1994)) and EcoRI (Ivanenko, et al., J. Biol. Chem. 379:459–465 (1998), Heitman and Model, Proteins 7:185–197 (1990), Heitman, Bioessays 14:445–454 (1992), Muir, et al., J. Mol. Biol. 274:722–737 (1997), Flores, et al., Gene 157:295–301 (1995)). Again, structure-based rational or semi-rational design approaches were employed with no absolute change of specificity reported. To date, a total of twelve structures of restriction enzymes have been determined (Pingoud and leltsch, Nucl. Acids Res. 29:3705–3727 (2001)). From these structures, it is becoming clear that substrate recognition does not adhere to a distinct set of rules. Consequently, the likelihood of engineering novel substrate specificities into existing endonucleases by purely rational design methods remains low. Furthermore, protein structure determination remains to be a costly and time-consuming endeavor.
Consequently, a rapid and more successful method of endonuclease engineering is required to identify amino acid substitutions responsible for altering substrate specificity without the requirement of protein structural information.